外泌体环状RNA作为结直肠癌潜在早期生物标志物及其机制研究

Colorectal cancer (CRC) is one of the most common digestive system tumors, which lacks early-detection non-invasive biomarker with high sensitivity and specificity. Recently, many researches and our previous findings have showed that the exosome circular RNA (circRNA), performing both the function transmission of exosome and the epigenetic regulation of circRNA, and having the features with stability, enrichment and easy detection, was expected to become a potential biomarker for CRC. The mechanism of exosome circRNA has not been investigated clearly. This study intends to screen the CRC-specific exosome circRNA based on the method of molecular epidemiology and the next generation high-throughput sequencing; and further to identify and evaluate the application value for exosome circRNA as CRC biomarker by the method of multi-stage validation and integration of clinical phenotypes. Furthermore, the method of cell and molecular biology are used to explore the competing endogenous RNA (ceRNA) effect of exosome circRNA on the CRC development and progression. All these will provide the basis for the determination of circRNA as a noninvasive biomarker for CRC screening, and afford important reference for the future prevention, intervention and treatment of CRC.

结直肠癌是最常见的消化系统恶性肿瘤之一，尚缺乏敏感、特异的无创性早期生物标志物。国内外研究和本课题组前期结果表明，外泌体环状RNA（circRNA）具有外泌体功能传递和circRNA表观遗传调控的双重特性，且具备稳定性高、含量丰富、易于检测等特点，有望成为结直肠癌潜在生物标志物。目前尚未见外泌体circRNA的作用机制研究。本课题拟基于分子流行病学研究方法，采用二代测序高通量方法，筛选结直肠癌特异的外泌体circRNA；运用多阶段人群样本验证，结合临床表型数据，鉴定具有结直肠癌早期生物标志物特性的外泌体circRNA，并初步评价其应用价值；结合细胞分子生物学实验方法和手段，探讨外泌体circRNA参与结直肠癌发生发展的竞争性内源RNA调控机制。以此为确定外泌体circRNA作为结直肠癌无创性早期筛查生物标志物奠定基础，并为今后结直肠癌的预防、干预和治疗提供重要参考。

外泌体环状RNA（circRNA）因具有高稳定性、高特异性等特性而作为肿瘤生物标志物的研究受到广泛关注。本研究通过整合结直肠癌组织（含癌旁）和外泌体circRNA等表达谱，筛选出3条结直肠癌差异性circRNAs。我们进一步在细胞本底及其培养液上清外泌体中检测候选circRNAs表达模式，发现circLPAR1在结直肠癌组织、肿瘤细胞本底以及外泌体中均显著低表达。随后，基于健康对照、结直肠息肉和结直肠癌病例人群，发现circLPAR1不受RNA酶、反复冻融、室温等外界因素影响，能稳定存在于人血浆外泌体中，且在结直肠癌病例中表达水平最低（P < 0.05）。通过受试者工作特征曲线（ROC）分析发现，外泌体circLPAR1具有作为结直肠癌早期诊断生物标志物的潜在能力（ROC曲线下面积AUC = 0.71, P < 0.05）。细胞恶性表型实验结果显示，circLPAR1能显著抑制结直肠癌细胞增殖、集落形成、侵袭与迁移等能力，而且可通过外泌体介导，实现细胞间功能传递，影响受体细胞的生物学活性。采用荧光原位杂交技术（FISH），发现circLPAR1在结直肠癌细胞质和细胞核中均有富集，为下游分子机制研究提供线索。进一步通过RNA-pull down技术，并整合蛋白组检测和生物信息学分析，我们发现circLPAR1能与EIF3H特异性结合，减少EIF3H与METTL3的相互作用，降低对癌基因BRD4 mRNA的环化能力，下调其表达水平，进而抑制结直肠癌的病理进程。综上所述，新型的外泌体circLPAR1在结直肠癌患者血浆中显著下调，可以较好地区分健康对照、结直肠息肉和结直肠癌患者，具有作为结直肠癌潜在的早期诊断的循环生物标志物。外泌体circLPAR1可能通过与METTL3竞争结合EIF3H，减少癌基因BRD4 的翻译，进而抑制结直肠癌的发生发展。